The endocannabinoid system consists of two cannabinoid receptors CB1 and CB2, which are G-protein coupled receptors and activated by mainly lipidic compounds. CB1 is a desirable therapeutic target due to its involvement in pathways related to addictive disorders and obesity. Hemopressin (HP), a nine-amino acid- peptide derived from the a chain of hemoglobin, has been shown to have selective inverse agonist activity against the CB1 receptor. In spite of hemopressin's tremendous potential as a safe and effective therapeutic, its further development has been hampered due to the variability of synthetic hemopressin in pharmacological assays. We hypothesize that the variability of hemopressin's effects in biological assays is due to the tendency of this peptide to form self-assembled nanostructures in solution under physiologically relevant conditions. Accordingly, we propose two specific aims: 1) To design and synthesize synthetic conjugates and analogs of hemopressin and conduct detailed nuclear magnetic resonance spectroscopy and transmission electron microscopy experiments to assess their aggregation and self-assembly properties. 2) To assess the affinity and efficacy of the newly designed conjugates and analogs of HP towards CB1 using four different assay systems: i) CB1 receptor binding assay, ii) [35S]GTPgS binding assay to monitor G-protein activation, iii) receptor-activated inhibition of cellular cAMP, and iv) b-arrestin recruitment. The central objective of this application is to evaluate the impact of hemopressin's ability to form nanofibrils on its pharmacological properties. Self-assembling biological peptides such as b-amyloid have profoundly improved our understanding of many aspects of neurobiology. Similarly, if we are able to show that self-assembly and aggregation of HP modulate its pharmacological activity, the finding will have promising therapeutic applicability in drug abuse research. PUBLIC HEALTH RELEVANCE: The endocannabinoid system refers to a group of neuromodulatory lipids and their receptors that are involved in a variety of biological processes, including addiction, appetite, memory, pain sensation, cognition and cancer cell proliferation. The endocannabinoid system consists of two cannabinoid receptors CB1 and CB2. Hemopressin is a hemoglobin-derived peptide that has been shown to act as a selective inverse agonist for the CB1 receptor, which is predominantly expressed in the motivational circuitry of the brain. CB1 is a desirable therapeutic target due to its involvement in pathways related to addictive disorders and obesity. Hemopressin is a potential drug candidate but its pharmacological activity has not been consistent and reproducible, possibly due to its propensity to form aggregates under physiological conditions. The goal of this study is to investigate the aggregation properties of hemopressin and develop analogs and/or conjugates of this peptide as therapeutic leads that show reproducible pharmacological profiles.